| Esophageal cancer is a common malignancy in the world.It has become one of the leading causes of death worldwide,and there are obvious regional and histological differences in the pathogenesis.According to data released in the National Cancer Registry,the incidence of esophageal cancer in China is the fourth highest incidence of malignant tumors and mortality,and it has become a major public health problem that threatens people's health.At present,it is considered that esophageal cancer is a disease with multiple factors,multi-gene participation,and multi-stage development.There are multiple pathways of oncogenes and tumor suppressor genes.Therefore,finding more effective esophageal cancer-related tumor markers and potential therapeutic targets for esophageal cancer has important practical significance for reducing the incidence and mortality of esophageal cancer.Drosophila Zeeste enhancer 2(enhancer of zeste homolog 2,EZH2)is an important member of the epigenetic regulatory factor Polycomb group(PcG)protein family,which together with SUZ12 and EED constitute PRC2 protein complex(core catalysis Subunits)have a highly conserved methyltransferase activity and are involved in epigenetics by silencing the thysine 27 th lysine(H3K27)trimethylation to induce silencing of a PRC2 target gene that has the ability to differentiate.Learning and regulation of gene expression.Melanoma-associated antigen(MAGE)is a cancerous testosterone antigen,which encodes a tumor rejection antigen that is processed in cells to produce an antigenic peptide that binds to human leucocyte antigene-I,HLA-I)is bound to form a complex and is recognized by autotoxic cytotoxicity T lymphocytes(CTL),which induce specific tumor cells to be specifically killed to achieve the purpose of treating tumors.Therefore,MAGE is an ideal target for CTL-mediated tumor-specific immunotherapy.More and moreexperiments show that some epigenetic mechanisms such as DNA methylation and histone methylation and acetylation play an important role in the regulation of MAGE-I gene expression.Previous studies have shown that DNA methylation and covalent modification of histones are involved in the silencing of various oncogenes and promote the development of cancer or human cancer.The most important mechanism is the silence of many cancer-associated genes through EZH2,and EZH2 can make the target gene promoterTrimethylation of histone H3 lysine K27 occurs.In this study,we explored the possible mechanisms of EZH2 promoting the progression of esophageal cancer and the apparent regulatory mechanisms of the tumor antigen MAGE-A11.We first used CCK-8 proliferation and colony formation assays to detect the effect of EZH2 on biological functions such as proliferation and cloning of esophageal cancer;and the interaction between EZH2 and MAGE-A11 was performed using chromatin immunoprecipitation assay.Research.The main research contents and results are as follows:Part one Effect of EZH2 gene on proliferation of human esophageal cancer cellsObjective: To investigate the effect of the overexpression or silence of EZH2 on the proliferation of esophageal cancer cells.Methods: Human esophageal cancer cell lines ECA109,TE1,KYSE30 and KYSE170 were selected as research objects.1)Real-time quantitative PCR(qPCR)and Western blotting were used to detect the expression of EZH2 mRNA and protein in esophageal cancer cells respectively.2)The expression of EZH2 mRNA in 4 esophageal cancer cells was detected by qPCR after overexpression and silencing of EZH2 gene.3)CCK-8 proliferation assay and clonogenic assay were used to detect the effect of overexpression and silencing of EZH2 gene and EZH2 inhibitor DZNep on esophageal cancer cell proliferation and colony formation.Result:1)The mRNA and protein levels of EZH2 in esophageal cancer ECA109 and TE1 cells were significantly higher than those in KYSE30 and KYSE170cells(P <0.05).2)The expression of EZH2 in esophageal cancer TE1 and ECA109 cells was down-regulated after transfected with EZH2-Sh RNA(all P <0.05),and the cell proliferation decreased(1.07 ± 0.08 vs 1.59 ± 0.09,P <0.05;0.88 ±0.08 vs 1.05 ± 0.11,P <0.05).The number of colony formation was down-regulated [(200.00 ± 11.43)vs(480.00 ± 13.10),P <0.05,(88.00 ± 8.16)vs(220.00 ± 14.69),P <0.05].3)EZH2 expression was increased in KYSE30 and KYSE170 cells transfected with EZH2 overexpression plasmid(all P<0.05),and cell proliferation was significantly increased(1.06 ± 0.07 vs 0.76 ± 0.06,P <0.05;3.36 ± 0.30 vs 1.50 ± 0.08,P <0.05).The number of colony formation was significantly higher than that of the control group(45.00 ± 3.27 vs 18.00 ±1.63,P <0.05,(65.00 ± 4.08)vs(23.00 ± 2.45,P <0.05].4)After DZNep treatment,the proliferation of ECA109 and TE1 cells decreased(t =21.29,16.83,all P<0.05)and the number of clone formation decreased [(200.00±6.53)vs(500.00±16.33),t=24.12,P<0.05;(80.00±10.88)vs(480.00±8.98),t=34.22,P<0.05].Summary:1)EZH2 mRNA and protein levels in esophageal cancer ECA109 and TE1 cells were significantly higher than those in KYSE30 and KYSE170 cells.2)knockdown of EZH2 gene expression or application of EZH2 inhibitor DZNep reduced the proliferation and colony formation rate of esophageal cancer;overexpression of EZH2 could effectively promote the proliferation and clonogenic capacity of esophageal cancer cells.Part two EZH2 regulates the expression of MAGE-A11 in esophageal cancer cellsObjective: To explore the effect of EZH2 on the expression of MAGE-A11 in esophageal cancer cells and the intrinsic mechanism of their interaction.Method:1)The expression of MAGE-A11 in each esophageal cancer cell line and the effect of demethylating agent DAC on the expression of MAGE-A11 were detected by real-time quantitative PCR(qPCR).2)The chromatin immunoprecipitation assay was used to analyze the transcriptional regulation effect of histone H3K27me3 on MAGE-A11 promoter in esophageal cancer cells.3)The effects of knockdown EZH2 on the expression of MAGE-A11 were detected by qPCR and Western Blot.4)Effect of Combined Use of DAC and DZNep on MAGE-A11 mRNA and Protein Levels in Eca109 and TE13 Esophageal Cancer Cells.Result:1)Eca109 and KYSE30 cells of esophageal carcinoma showed relatively low expression of MAGE-A11 mRNA(P<0.05),while TE13 cells showed relatively high expression of MAGE-A11 mRNA(P<0.05).2)Demethylating agent DAC induced significant MAGE-A11 induction in MAGE-A11-overexpressing Eca109 and KYSE30 cells(P<0.05),whereas only DAC pairs were detected in MAGE-A11-expressing TE13 cells.Mild induction of MAGE-A11.3)Silencing markers on the promoter of MAGE-A11 in Eca109 cells accounted for more,such as H3K27me3,H3K9me3;and the activation marker H3K4me3 accounted for less.Conversely,the proportion of silent markers on the MAGE-A11 promoter decreased in TE13 cells,such as H3K27me3,H3K9me3,whereas the proportion of activation markers H3K4me3 increased.Treatment of Eca109 cells with DAC reduced the binding of H3K27me3 and H3K9me3 to the MAGE-A11 promoter,whereas the binding of H3K4me3 to the MAGE-A11 promoter increased.4)After knockdown of EZH2 or treatment with DZNep in Eca109 cells,the expression of H3K27me3 on the MAGE-A11 promoter was significantly decreased.5)After knockdown of EZH2 expression,the expression of MAGE-A11 in Eca109 cells was significantly increased.6)In MAGE-A11 hypermethylated Eca109 cells,the combination of DZNep and DAC increased the expression of MAGE-A11 mRNA,which was significantly higher than that of DZNep alone;while in MAGE-A11 hypomethylated TE13 cells,DZNep and The combination of DAC or DZNep alone had no significant effect on MAGE-A11 mRNA expression.Summary:1)Eca109 and KYSE30 cells of esophageal cancer show MAGE-A11 hypermethylation status;TE13 is in MAGE-A11 hypomethylation state.2)In hypermethylated Eca109 cells,EZH2 participates in transcriptional regulation of the MAGE-A11 promoter by mediating histone H3K27 me.Conclusion:EZH2 can promote the proliferation and colony formation of esophageal cancer cells,and EZH2-mediated histone H3K27me3 methylation participates in transcriptional regulation of the MAGE-A11 promoter in MAGE-A11 hypermethylated esophageal cancer cells.The abnormal overexpression of EZH2 can serve as a predictor of poor prognosis in esophageal cancer patients,and block the expression of EZH2,which may provide a new strategy for ESCC treatment. |
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